1. Field of the Invention
The present invention relates to sinusoidal voltammetry with either lock-in detection or Fourier Transform based computer methods as an electroanalytical method for making fast, very small volume chemical analyses of a fluid.
2. Description of the Prior Art
Several electroanalytical techniques have been developed for measurement of small volumes of fluid in short time periods, such as a few hundred milliseconds. For example, fast scan voltammetry is described by W. G. Kuhr et.al., Brain Res. 381 at 168-71 (1986). Chronoamperometry is described by A. G. Ewing et.al., Brain Res. 249 at 361-70 (1982). DC amperometry is described by D. J. Leszczyszyn et al., J. Biol. Chem., 265 at 1473-7 (1990). These methods have been used for the measurement of various electroactive neurotransmitters, such as dopamine, norepinephrine and serotonin, to ascertain the kinetics of release and uptake process in vivo in the mammalian brain on a subsecond time scale.
In particular, electrochemical detection at carbon fiber microelectrodes with fast cyclic voltammetry has been used successfully for the characterization of dopamine release and uptake in vivo as described by R. M. Wightman, Anal. Chem. 53 at 1125a-30a (1981) and W. G. Kuhr et.al., Brain Res. 381 at 168-71 (1986). The fast scan rates, i.e. 100 to 300 volts per second, provide excellent temporal resolution and enhanced selectivity over compounds that have slower electron transfer kinetics than the analyte of interest. The use of Nation coated microelectrodes has improved the limits of detection to the order of 50 nM for dopamine when used in combination with signal processing and sampling methods.
Additional improvements in sensitivity in fast scan cyclic voltammetry have been gained through improvement to signal processing and sampling strategies. While voltammetry has also been used to monitor secretion of catecholamines from individual cells on millisecond time scales, DC amperometric recordings have been found to give better signal quality. Similar trends are found when electrochemical detection was used in combination with separation methods, e.g. HPLC, capillary LC and capillary electrophoresis. DC methods generally have better sensitivity because of the bandpass of the measurement is narrower, that is low pass filtering reduces the high frequency noise and there is a virtual absence of background currents.
Traditionally, electrochemical methods generally improve the signal-to-noise ratio by discriminating the faradaic signal from the background components in the time domain through application of pulsed waveforms, i.e. differential pulse polarography and square wave voltammetry. Pulse methods are able to discriminate the faradaic current from the charging current in the time domain. Since charging currents decay much more rapidly than faradaic current, i.e. exponentially as compared to the inverse square root. However, the analytical faradaic current is not totally discriminated from the charging current and most of the signal is discarded because sampling must be done late in the pulse cycle. Even under these conditions, limits of detection on the order of 10.sup.-8 M can be obtained with pulsed methods, albeit at the cost of analysis time, typically at 10 to 100 seconds per scan.
Alternatively, modulation techniques have been used to great effect in a number of circumstances to improve signal-to-noise ratios. In these techniques, the signal is imposed on a modulated carrier wave, such as a sine wave. The modulation frequency can be chosen to move a signal into a region of the frequency spectrum where there are minimal noise contributions. This is particularly useful for detection of species in real time on a subsecond time scale where 1/f noise is often a problem. Often, a lock-in amplifier is used in modulation methods to decrease the bandwidth of the monitored signal to discriminate the signal from the noise on the basis of frequency and phase. The decreased bandwidth serves to reduce noise, enhance signal recognition and increase the signal-to-noise ratio.
The frequency domain has only been used in a few electroanalytical techniques to enhance the signal-to-noise ratios in electrochemical analysis. In AC voltammetry, a potential ramp is applied to the electrode, typically 10 to 15 millivolts per second, and a small amplitude sine wave, typically less than 50 millivolts, usually on the order of 10 millivolts, is superimposed onto the linear ramp. Measurement of the fundamental and harmonic frequencies are taken using a lock-in amplifier. Small amplitude modulations are used to minimize the nonlinear effects and enhance resolution. The scan time is determined by the slope of the linear ramp. The time of analysis typically various from 20 to 200 seconds per scan. Since the potential is modulated at 100 to 1,000 times the fundamental frequency of the ramp, modulation frequencies are typically on the order of ten to hundreds of Hertz. See for example, Eccles, Crit. Rev. Anal. Chem. 22 at 345-80 (1991).
While small amplitude modulation works well at a large electrode at conventional scan rates, that is electrodes of millimeter dimensions with scan rates in the range of 10 to 50 millivolts per second with a corresponding time analysis of 20 to 200 seconds, it would be difficult to implement this type of modulation strategy at scan rates fast enough to provide information relevant to neurotransmission. For example, the measurement of stimulated dopamine released in a rat brain, scan rates of 300 volts per second are commonly employed with a complete scan obtained in less than 10 milliseconds. Thus, to maintain adequate potential resolution in AC voltammetry, one would have to use the modulation frequency in excess of 100 kHz. Similarly, it would be difficult to use pulse methods to make a complete voltammetric measurement on a millisecond time scale. Either the period of the step would be too short to allow discrimination against charging current, e.g. 10 microseconds per step, or the number of potential steps must be reduced which would limit resolution.
There are no neurochemical molecules which have electron transfer kinetics fast enough to allow this modulation frequency. Therefore, any methods requiring multiple potential steps, such as chronoamperometry, pulse voltammetry or square wave voltammetry or potential modulation such as AC voltammetry, will be difficult to implement to measure these dynamic chemical phenomena.
Sinusoidal voltammetry, an analog of continuous scan cyclic voltammetry, uses a large amplitude sinusoid exclusively as the potential waveform. An analog of this method was originally called "oscillographic polarography" and predates the use of linear scan techniques as currently used in cyclic voltammetry. See M. Heyrovsky et al., in Electroanalytical Chemistry: A Series of Advancements, Marcel Dekker, Inc., New York, Volume 2 at 193-56 (1967). The use of a triangular wave in a cyclic voltammetric experiment gained favor over the use of a sine wave because of the theoretical complications imposed by the fact that in the case of a sine wave the scan rate is continuously changing throughout the duration of the experiment. While the experimental equivalent to steady state sinusoidal voltammetry has been mathematically described, little work has been done in this field over the last 20 years. See A. E. Remick et al., J. Electrochem. Soc. 109 at 628-34 (1962).
A sinusoidal waveform has been used to simplify digital filtering in the frequency domain for voltammetric analysis. See J. T. Long, Electroanalysis 4 at 429-37 (1992). It was found that the sinusoidal waveform produced better signal-to-noise ratios when using digital filter routines in flow injection analysis experiments. Fourier transform methods have been used to examine many of the properties of different electroanalytical experiments, but the analytical advantage of this technique has not been exploited.
Most electrochemical methods rely on differences between the formal potential (half widths) of compounds present in a sample to generate the selectivity for measurement which leads to an effective resolution of only 6 to 10 components. In practice, this has severely limited the utility of electrochemical methods for the analysis of many complex matrices. Additional selectivity could be obtained only through the use of other electrochemical methods to clean up the sample prior to electroanalysis or the addition of ion-selective membranes to the electrode surface to alter the transport of the sample constituents to the electrode surface. What is needed then is a method for using large amplitude sinusoidal waveform in fast electroanalytical tests.
Therefore what is needed is some methodology which can exploit the vast diversity in electron transfer rates observable at solid electrodes to obtain additional selectivity in the electrochemical measurement.